9α-Aza-9α-homoerythromycin compounds, pharmaceutical composition and therapeutic method

ABSTRACT

Certain novel 9-deoxo-4&#34;-deoxy-4&#34;-amino-9a-aza-9a-homoerythromycin A derivatives; a method of treating a bacterial infection in a mammalian subject using these novel erythromycin A derivatives; pharmaceutical compositions containing the novel erythromycin A derivatives; and intermediates and processes for making the novel erythromycin A derivatives.

BACKGROUND OF THE INVENTION

This invention relates to novel chemical compounds which haveantibacterial activity, and which are useful in the chemotherapy ofbacterial infections in mammalian subjects. More particularly, the novelantibacterial agents of this invention are derivatives of the well-knownmacrolide antibiotic, erythromycin A, the compound of the followingchemical structure: ##STR1## Even more particularly, the novelantibacterial agents of this invention are derivatives or erythromycinA, in which the 14-membered lactone ring has been expanded to a15-membered ring by insertion of a nitrogen atom between ring-members 9and 10, and the 4"-alpha-hydroxy group has been replaced by asubstituent bonded to the 4"-position through a nitrogen atom (e.g. aprimary amino group).

Thus the antibacterial agents of this invention can be regarded asderivatives of the compound of the formula II, viz.: ##STR2## and forthe purposes of this specification, the structure II is named chemicallyas 9a-aza-9a-homoerythromycin A, i.e. the locant 9a is used to identifythe additional ring member in the lactone ring.

9a-Aza-9a-homoerythromycin A compounds have been disclosed in publishedBritish patent application No. 2,094,293 and U.S. Pat. No. 4,328,334,where they are named as 11-aza-10-deoxo-10-dihydroerythromycin Acompounds. 4"-Deoxy-4"-amino-erythromycin A antibacterial agents areknown from U.S. Pat. No. 4,150,220.

SUMMARY OF THE INVENTION

This invention provides novel macrolide antibiotics of the formula:##STR3## and the pharmaceutically-acceptable acid-addition saltsthereof; wherein

R¹ is selected from the group consisting of hydrogen and methyl; and

R² and R³ are each selected from the group consisting of hydrogen andamino;

provided that R² and R³ are always different.

Additionally, this invention provides a method of treating a bacterialinfection in a mammalian subject, which comprises administering to saidsubject an anti-bacterially effective amount of a compound of formulaIII, wherein R¹, R² and R³ are as defined above; and also pharmaceuticalcompositions containing a compound of formula III, wherein R¹, R² and R³are as defined above.

Yet further, this invention provides novel compounds of the formula:##STR4## and the acid-addition salts thereof, wherein R⁴ is selectedfrom the group consisting of hydrogen, acetyl and propionyl. Thecompounds of formula IV are useful as intermediates to the antibacterialagents of formula III, wherein R¹, R² and R³ are as defined previously.

Preferred compounds of the formula III are:

9-deoxo-9a-methyl-4"-deoxy-4"-alpha-amino-9a-aza-9a-homoerythromycin A(the compound of formula III, wherein R¹ is methyl, R² is amino and R³is hydrogen); and

9-deoxo-9a-methyl-4"-deoxy-4"-beta-amino-9a-aza-9a-homoerythromycin A(the compound of formula III, wherein R¹ is methyl, R² is hydrogen andR³ is amino).

A particularly useful intermediate of the formula IV is:

9-deoxo-9a-methyl-4"-deoxy-4"-oxo-9a-aza-9a-homoerythromycin A (thecompound of formula IV, wherein R⁴ is hydrogen).

Further compounds which show antibacterial activity can be obtained byacylation of the group R² or R³ in a compound of formula III, when saidR² or R³ is amino. In this connection, acyl derivatives can be preparedby acylation of said R² or R³ as amino using a variety of carboxylicacids of the formula R⁵ --CO--OH and sulfonic acids of the formula R⁶--SO₂ --OH. The acylated derivatives so formed are also to be consideredpart of this invention.

DETAILED DESCRIPTION OF THE INVENTION

The antibacterial agents of this invention are of formula III, whereinR¹ is hydrogen or methyl, and R² and R³ are hydrogen or amino, providedthat R² and R³ are always different. These compounds can be preparedeasily and conveniently from a 4"-deoxy-4"-aminoerythromycin Aderivative of the formula V: ##STR5## wherein R² and R³ are eachhydrogen or amino, provided R² and R³ are always different. This iscarried out according to Scheme A, in which only partial structures areshown. ##STR6##

In the first step of Scheme A, the 4"-deoxy-4"-amino-erythromycin Acompound of formula V is converted into its oxime of formula VI. This isusually carried out by treating the compound of formula V with an excessof hydroxylamine, or an acid-addition salt thereof (e.g. thehydrochloride), in pyridine solution, at a temperature in the range from20° to 60° C. The reaction usually takes several hours, e.g. about 15 to50 hours, to reach completion, after which the product is isolated bydiluting the reaction mixture with water, and extracting the productinto a volatile, water-immiscible, organic solvent, such as ether orethyl acetate. The product can then be recovered by drying the organicsolvent, followed by evaporating the organic solvent in vacuo.

In the second step of Scheme A, the oxime of the formula VI is subjectedto a Beckmann rearrangement to give the ring-expanded,9a-aza-9a-homo-amide of the formula VII. This ring-expansion isconveniently carried out by treating the oxime of formula VI with anexcess of 4-toluenesulfonyl chloride in the presence of a base at aboutroom temperature. In one method, the oxime is added to aqueous acetonecontaining sodium bicarbonate, and then the 4-toluenesulfonyl chlorideis added with the pH being maintained at about 8 by the addition ofsodium hydroxide solution. Alternatively, the rearrangement can becarried out in a water-immiscible organic solvent, such as chloroform,in which case a slight excess of a tertiary amine is added to the oximeand the 4-toluenesulfonyl chloride. The rearrangement proceeds quiterapidly at ambient temperature, and in practice the reaction is allowedto proceed without external cooling. Under these conditions it isnormally complete within 1 to 2 hours. When aqueous acetone has beenused as the reaction solvent, the reaction mixture is diluted with waterand the product is extracted into a volatile, water-immiscible organicsolvent at a basic pH, followed by solvent evaporation. When awater-immiscible, organic solvent has been used for the Beckmannrearrangement, the product is extracted into an aqueous phase byextraction with water at an acidic pH. The water extract is thenbasified and the product is extracted into a volatile, water-immiscibleorganic solvent. Finally the water-immiscible, organic solvent is driedand evaporated in vacuo to give the desired 9a-aza-9a-homo-amide of theformula VII.

The compounds of the formula III, wherein R¹ is hydrogen, can beobtained by reduction of the 9,9a-amide grouping in the compound offormula VII. This can be carried out using a variety of agents known toreduce amides to amines, but in the present instance a particularlyconvenient reducing agent is sodium borohydride.

When the 9a-aza-9a-homo-amide of the formula VII is reduced to thecorresponding amine of formula III, wherein R¹ is hydrogen, a solutionof the starting amide in a lower-alkanol, e.g. methanol, is treated withan excess of solid sodium borohydride, at a temperature from 0° to 30°C., and usually at about room temperature. At room temperature, thereaction proceeds quite smoothly and quickly, and it is normallycomplete within 1 to 2 hours. The reaction mixture can then be dilutedwith water and a volatile, water-immiscible, organic solvent, e.g. ethylacetate. The pH is raised to about 10, and the organic layer is removedand dried. Evaporation of the organic layer then affords the desiredcompound of formula III, wherein R¹ is hydrogen.

The compounds of formula III, wherein R¹ is methyl, can be prepared bymethylation at N-9a of the corresponding compound of formula III,wherein R¹ is hydrogen. However, before methylating at N-9a, it ispreferable to protect the amino group at C-4", since the latter group isalso susceptible to methylation. Thus, the preferred method ofconverting a compound of formula III, wherein R¹ is hydrogen, into thecorresponding compound of formula III, wherein R¹ is methyl, involvesprotection at C-4", followed by methylation at N-9a, followed bedeprotection at C-4".

A variety of amino protecting groups can be used to protect the primaryamino function at C-4", but particularly convenient groups are thebenzyloxycarbonyl group and the 4-nitrobenzyloxycarbonyl group. Thesegroups are attached to C-4" and they are removed from C-4" by standardmethods, well-known in the art.

For example, the compound of formula III, wherein R¹ is methyl, istreated with a slight excess of benzyloxycarbonyl chloride or4-nitrobenzyloxycarbonyl chloride in the presence of a tertiary amine,such as pyridine or triethylamine, at room temperature, in areaction-inert organic solvent. The reaction proceeds quite rapidly, andit is usually complete within one hour. When a water-immiscible solvent,such as chloroform, has been used, the product is extracted into waterat an acidic pH (e.g. pH 2) and then it is back-extracted into avolatile, water-immiscible, organic solvent at a basic pH (e.g. pH 10).Evaporation of the solvent then affords the C-4"-protected-aminocompound. When a water-immiscible solvent has been used, the product canbe isolated by diluting the reaction mixture with water and extractingwith a volatile, water-immiscible organic solvent at a basic pH (e.g. pH10). Evaporation of the solvent affords the product.

Methylation of the C-4"-protected-amino compound of the formula III,wherein R¹ is hydrogen, can be carried out conveniently using an excessof formaldehyde and formic acid in a reaction-inert organic solvent,such as chloroform. The reaction is usually carried out at a temperaturefrom 60° to 100° C., and it usually takes a few hours, e.g. 2 to 6hours, to reach completion. At the end of the reaction, the reactionmixture is cooled, and the C-4"-protected-amino compound of formula III,wherein R¹ is methyl, is isolated in exactly the same manner asdescribed for isolation of the protected compound of formula III,wherein R¹ is hydrogen.

The benzyloxycarbonyl or 4-nitrobenzyloxycarbonyl protecting group canbe removed from the amino at C-4" by hydrogenolysis in glacial aceticacid solution using palladium-on-carbon catalyst, according to standardprocedures. The reaction is usually carried out at room temperature, andat a hydrogen pressure of from 1 to 10 kg/cm². The reaction is normallycomplete within a few hours, e.g. 4 to 10 hours. The catalyst is thenremoved by filtration and the acetic acid solution of the product ispartitioned between water and a volatile, water-immiscible, organicsolvent, such as ethyl acetate, at pH 8 to 10. The organic layer isremoved, dried and evaporated to give the desired compound of formulaIII, wherein R¹ is methyl.

The novel 9a-aza-9a-homoerythromycin A derivatives of formula III,wherein R¹ is methyl and R² and R³ are as defined previously, can alsobe prepared from the known 9a-aza-9a-homoerythromycin A derivative ofthe formula VIII: ##STR7## For preparation of the compounds of formulaIII, wherein R¹ is methyl, and R² and R³ are as defined previously, thecompound of formula VIII is first converted into the corresponding4"-keto compound of the formula IX: ##STR8## Conversion of the compoundof the formula VIII into the compound of the formula IX involvesprotection of the 2'-hydroxy group, followed by oxidation of the4"-hydroxy group, followed by removal of the protecting group from the2'-hydroxy group.

Protection of the 2'-hydroxy group is usually achieved using alower-alkanoyl protecting group, e.g. acetyl or propionyl. The acetyl orpropionyl group is attached to the 2'-hydroxy group by treatment of thecompound of formula VIII with a slight excess of acetic anhydride orpropionic anhydride in chloroform, at room temperature, for severalhours, according to standard procedures. Oxidation to the corresponding2'-O-acetyl-4"-deoxy-4"-keto compound (or its 2'-O-propionyl analog) isthen achieved by treatment with dimethyl sulfoxide and a carbodiimide inthe presence of a base. N-Ethyl-N'-(N,N-dimethylaminopropyl)carbodiimideis conveniently used as the carbodiimide and pyridinium trifluoroacetateis conveniently used as the base. Finally, removal of the 2'-O-acetyl or2'-O-propionyl group can be achieved by solvolysis with methanol at 10°to 30° C. for 1 to 2 days, followed by removal of the methanol byevaporation in vacuo.

For preparation of the compound of the formula III, wherein R¹ ismethyl, R² is amino and R³ is hydrogen, the compound of formula IX isconverted into its C-4" oxime, and then the oxime is reduced withgaseous hydrogen over a Raney nickel catalyst.

The oxime is prepared by treating the ketone (IX) with an excess ofhydroxylamine hydrochloride in methanol solution at room temperature forseveral hours. It is then isolated by removal of the solvent in vacuo.Reduction of the C-4" oxime of the compound of the formula IX isachieved by treatment with hydrogen, at room temperature, over a Raneynickel catalyst, in a solvent such as a lower-alkanol (e.g. ethanol) andat a pressure in the range from 1 to 10 kg/cm², and preferably 4 to 5kg/cm². The catalyst is removed by filtration and the product can thenbe recovered by solvent evaporation.

For preparation of the compound of formula III, wherein R¹ is methyl, R²is hydrogen and R³ is amino, the compound of formula IX can bereductively aminated. This can be achieved by contacting the compound offormula IX with an excess of ammonium acetate, in a lower-alkanol, suchas methanol, and then reducing the resulting adduct with sodiumcyanoborohydride. In practice this produces the compound of formula III,wherein R¹ is methyl, R² is hydrogen and R³ is amino, together with itsC-4"-epimer. Also, some of the corresponding C-4"-hydroxy compounds(compound VIII and its C-4"-epimer) are formed. The C-4"-hydroxycompounds are readily removed during work-up. The total reaction productis partitioned between ethyl acetate and water at pH 6, under whichconditions the C-4"-hydroxy compounds are extracted into the organiclayer while the C-4'-amino compounds remain in the aqueous layer. Atthis point, the ethyl acetate layer is separated and discarded, and thepH of the aqueous layer is raised to 9 to 10. The C-4"-amino compoundscan then be extracted into an organic phase, which is then separated,dried and evaporated in vacuo. The compound of formula III, wherein R¹is methyl, R² is hydrogen and R³ is amino, can be separated from itsC-4"-epimer by chromatography.

The antibacterial agents of this invention of formula III, and theintermediate compounds of formulae IV, V, VI, VII and IX, can all bepurified after preparation, if desired, by standard procedures formacrolide compounds. Such procedures recrystallization, columnchromatography, preparative thin-layer chromatography andcounter-current distribution.

The antibacterial compounds of the formula III, and the intermediates offormula IV, are basic and therefore they will form acid-addition salts.All such salts are within the scope of this invention, and they can beprepared by standard procedures for macrolide compounds. The compoundsof formula III and IV contain more than one basic center, and mono-, di-or tri-acid-addition salts can be prepared. For di- andtri-acid-addition salts the anionic counter ions can be the same ordifferent. In general, for preparation of the acid-addition salts, thecompound of formula III or IV is combined with a stoichiometric amountof an appropriate acid in an inert solvent, and then the salt isrecovered by solvent evaporation, by filtration if the salt precipitatesspontaneously, or by precipitation using a non-solvent followed byfiltration. Typical salts which can be prepared include sulfate,hydrochloride, hydrobromide, nitrate, phosphate, citrate, tartrate,pamoate, sulfosalicylate, methanesulfonate, benzenesulfonate and4-toluenesulfonate salts.

The starting materials of formula V, wherein R² and R³ are each hydrogenor amino, provided R² and R³ are always different, can be prepared byreductive amination of 4"-deoxy-4"-oxo-erythromycin A, the compound ofthe formula X: ##STR9##

In order to prepare the compound of formula V, wherein R² is hydrogenand R³ is amino, a mixture of the compound of formula X and an excess ofammonium acetate in methanol is hydrogenated at ambient temperature, ata pressure of about 4 kg/cm², in the presence of 10% palladium-on-carboncatalyst. This affords predominantly the C-4"-beta-amino compound, whichcan be obtained pure by trituration under ether.

The compound of formula V, wherein R² is amino and R³ is hydrogen, canbe prepared by hydrogenation of a mixture of the compound of formula Xand an excess of ammonium acetate in methanol, at ambient temperature,at a hydrogen pressure of ca. 4 kg/cm², using a Raney nickel catalyst.This affords predominantly the C-4"-alpha-amino compound, which can beobtained pure by repeated recrystallization from a solvent such asisopropanol.

4"-Deoxy-4"-oxo-erythromycin A, the compound of formula X, can beprepared according to the procedures described in U.S. Pat. No.4,150,220.

9-Deoxo-9a-methyl-9a-aza-9a-homoerythromycin A, the compound of formulaVIII, can be prepared according to the procedure of published Britishpatent application No. 2,094,293. See further Example 1 of said GB No.2,094,293, wherein the compound of formula VIII is namedN-methyl-11-aza-10-deoxo-10-dihydro-erythromycin A. See also U.S. Pat.No. 4,328,344 and West German Offenlegungsschrift DE-OS No. 3012533.

The compounds of formula III, wherein R¹, R² and R³ are as definedpreviously, are useful as antibacterial agents both in vitro and invivo, and their spectrum of activity is similar to that of erythromycinA. Consequently, they can be used for the same purposes, and in the samemanner, as erythromycin A.

In general, the compounds of formula III, wherein R¹, R² and R³ are asdefined previously, and salts thereof, exhibit in vitro activity againsta variety of Gram-positive microorganisms, e.g., Staphylococcus aureusand Streptococcus pyogenes, and against certain Gram-negativemicroorganisms such as those of spherical or ellipsoidal shape (cocci).Their activity is readily demonstrated by in vitro tests against variousmicroorganisms in a brain-heart infusion medium by the usual two-foldserial dilution technique. Their in vitro activity renders them usefulfor topical application; for sterilization purposes, e.g., sick-roomutensils; and as industrial antimicrobials, for example, in watertreatment, slime control, paint and wood preservation. For in vitro usefor topical application, it will usually be convenient to preparepharmaceutical compositions, in which the compound of formula III iscombined with a pharmaceutically-acceptable carrier or diluent, forexample in the form of ointments and creams. Appropriate carriers anddiluents for these purposes include mineral oils and vegetable oils, andsolvents such as water, alcohols, and glycols, and mixtures thereof.Such a pharmaceutical composition will normally contain thepharmaceutically-acceptable carrier and the compound of formula III in aweight ratio in the range from 4:1 to 1:4.

Additionally the compounds of formula III, wherein R¹, R² and R³ are asdefined previously, and the pharmaceutically-acceptable salts thereof,are active in vivo versus a variety of Gram-positive microorganisms,e.g. Staphylococcus aureus and Streptococcus pyogenes, and also certainGram-negative microorganisms, via the oral and parenteral routes ofadministration, in animals, including man. Their in vivo activity ismore limited than their in vitro activity as regards susceptibleorganisms, and it is determined by the usual procedure which comprisesinfecting mice of substantially uniform weight with the test organismand subsequently treating them orally or subcutaneously with the testcompound. In practice, the mice, e.g., 10, are given an intraperitonealinoculation of suitable diluted cultures containing approximately 1 to10 times the LD₁₀₀ (the lowest concentration of organisms required toproduce 100% deaths). Control tests are simultaneously run in which micereceive inoculum of lower dilutions as a check on possible variation invirulence of the test organism. The test compound is administered 0.5hour post-inoculation, and is repeated 4, 24 and 48 hours later.Surviving mice are held for four days after the last treatment and thenumber of survivors is noted.

When used in vivo to treat a bacterial infection in a mammalian subject,especially man, the compounds of formula III and salts thereof can beadministered alone, or, preferably, in the form of pharmaceuticalcompositions containing a pharmaceutically-acceptable carrier ordiluent. Such compositions can be administered orally, for example astablets or capsules, or parenterally, which includes subcutaneous andintramuscular injection. The pharmaceutically-acceptable carrier willdepend on the intended mode of administration. For example, lactose,sodium citrate and salts of phosphoric acid, together withdisintegrating agents (such as starch) and lubricating agents (such asmagnesium stearate, sodium lauryl sulfate and talc) can be used as thepharmaceutically-acceptable carrier in tablets. Also, for use incapsules useful pharmaceutically-acceptable carriers are lactose andhigh molecular weight polyethylene glycols (e.g., having molecularweights from 2,000 to 4,000). For parenteral use, sterile solutions orsuspensions can be prepared, wherein the pharmaceutically-acceptablecarrier is aqueous (e.g., water, isotonic saline or isotonic dextrose)or non-aqueous (e.g., fatty oils of vegetable origin such as cottonseedor peanut oil, or polyols such as glycerol or propylene glycol).

For in vivo use of a compound of the formula III, or a salt thereof, apharmaceutical composition will usually contain thepharmaceutically-acceptable carrier and the compound of formula III orsalt thereof in a weight ratio in the range from 4:1 to 1:4.

When used in vivo to treat a bacterial infection in a mammalian subject,either orally or parenterally, the usual daily dosage will be in therange from 5 to 100 mg/kg of body weight, especially 10 to 50 mg/kg ofbody weight, in single or divided doses.

The following examples and preparations are being provided solely forthe purpose of additional illustration. Proton nuclear magneticresonance spectra (¹ H-NMR spectra) were measured as solutions indeuterated chloroform (CDCl₃), and peak positions of diagnosticabsorptions are reported in parts per million (ppm) downfield frominternal tetramethylsilane.

EXAMPLE 19-Deoxo-9a-methyl-4"-deoxy-4"-beta-amino-9a-aza-9a-homoerythromycin A A.9-Deoxo-4"-deoxy-4"-beta-benzyloxycarbonylamino-9a-aza-9a-homoerythromycinA

To a stirred solution of 0.5 g (0.68 mmole) of9-deoxo-4"-deoxy-4"-beta-amino-9a-aza-9-homoerythromycin A and 0.11 ml(1.0 mmole) of pyridine in 20 ml of dichloromethane was added a solutionof 0.15 ml (1.0 mmole) of benzyloxycarbonyl chloride (benzylchloroformate) in 5 ml of dichloromethane. The reaction mixture wasstirred at room temperature for 30 minutes, and then an excess of waterwas added. The pH of the aqueous phase was adjusted to 2, and theorganic layer was removed and discarded. The pH of the aqueous layer wasraised to 5, and then the aqueous layer was extracted with chloroformand the chloroform extracts were discarded. The pH of the aqueous layerwas then raised to 8 and the aqueous layer was again extracted withchloroform. The latter chloroform extracts were washed with water,dried, and evaporated in vacuo to give 0.43 g of the4"-beta-benzyloxycarbonylamino derivative.

B. 9-Deoxo-9a-methyl-4"-deoxy-4"-beta-benzyloxycarbonylamino-9a-aza-9a-homoerythromycin A

A mixture of the product of Part A (0.43 g), 0.2 ml of 37% aqueousformaldehyde, 0.05 ml of 98% formic acid and 30 ml of chloroform washeated under reflux for 3.5 hours. The reaction mixture was then cooledand diluted with an excess of water. The pH was adjusted to 9, and themixture was extracted with chloroform. The chloroform extracts werewashed with water, dried, and evaporated in vacuo to give 0.40 g of the9a-methyl derivative.

C. 9-Deoxo-9a-methyl-4"-deoxy-4"-beta-amino-9a-aza-9a-homoerythromycin A

The product of Part B (0.40 g) was dissolved in 5 ml of glacial aceticacid, and then 100 mg of 10% palladium-on-carbon was added, undernitrogen. The resulting mixture was shaken under an atmosphere ofhydrogen for 5.5 hours at a pressure of ca. 4 kg/cm², and then thecatalyst was removed by filtration. To the filtrate was added ethylacetate and water and the pH of the aqueous phase was adjusted to 9.5.The ethyl acetate layer was removed, and the aqueous layer was extractedwith further ethyl acetate. The ethyl acetate solutions were combined,washed with water and dried. Evaporation of the dried solution gave 0.04g of 9-deoxy-9a-methyl-4"-deoxy-4"-beta-amino-9a-aza-9a-homoerythromycinA.

The ¹ H-NMR spectrum of the product showed absorptions at 2.13 (broadsinglet, 9H) and 3.28 (singlet, 3H) ppm.

EXAMPLE 29-Deoxo-9a-methyl-4"-deoxy-4"-alpha-amino-9a-aza-9a-homoerythromycin A

The title compound can be prepared from9-deoxo-4"-deoxy-4"-alpha-amino-9a-aza-9a-homoerythromycin A by reactionwith benzyloxycarbonyl chloride, followed by reaction withformaldehyde-formic acid, followed by hydrogenolysis, using theprocedures of Parts A, B and C of Example 1.

EXAMPLE 3 9-Deoxo-4"-deoxy-4"-alpha-amino-9a-aza-9a-homoerythromycin AA. 4"-Deoxy-4"-alpha-amino-erythromycin A oxime

A mixture of 6.4 g (8.6 mmole) of 4"-deoxy-4"-alpha-amino-erythromycinA, 3.2 g (46 mmole) of hydroxylamine hydrochloride and 65 ml of pyridinewas heated to 50° C., and maintained at that temperature for ca. 18hours. The reaction mixture was then added to a mixture of diethyl etherand water, and the pH of the aqueous layer was adjusted to 10. Thelayers were separated, and the aqueous layer was extracted further withether. The combined ether solutions were washed with water, followed bysaturated sodium chloride solution, and then the ether solution wasdried (Na₂ SO₄). Evaporation of the ether solution gave a foam. To thefoam was added more ether, and the mixture was heated on a steam bathand then allowed to cool. The solid material was filtered off to give afirst crop (3.86 g) of the required oxime. The ether filtrate wasevaporated in vacuo to give a second crop (1.9 g) of the required oxime.

B. 4"-Deoxy-4" -alpha-amino-9a-aza-9a -homoerythromycin A

To a solution of the second crop of the oxime of Part A (1.9 g; 2.5mmole) in a mixture of 40 ml of acetone and 30 ml of water was added 1.5g (18 mmole) of sodium bicarbonate, and then the mixture was cooled toice-bath temperature. To the resulting mixture was added a solution of1.0 g (5.0 mmole) of 4-toluenesulfonyl chloride in ca. 10 ml of acetone,dropwise with stirring during 10 minutes. During the addition, aqueoussodium hydroxide was added as needed to keep the pH at approximately 8.Stirring was continued for 30 minutes, and then the reaction mixture waspoured into a mixture of water and dichloromethane. The pH of theaqueous layer was adjusted to 5 and the dichloromethane layer wasremoved. The aqueous residue was then extracted with dichloromethane atpH 6.0 and 10. The dichloromethane solution from the extraction at pH 10was evaporated in vacuo and the residue recrystallized from ether, togive a first crop of the desired 9a-aza-9a -homo compound. Thedichloromethane solution from the extraction at pH 6.5 was evaporated invacuo to give a second crop of the desired 9a -aza-9a-homo compound. Themother liquor from the ether recrystallization was evaporated in vacuoto give a third crop of the desired 9a -aza-9a-homo compound. The threecrops were combined to give 1.0 g of the desired 9a-aza-9a-homocompound.

C. 9-Deoxo-4"-deoxy-4"-alpha-amino-9a -aza-9a-homoerythromycin A

A solution of the product of Part B (1.0 g; 1.3 mmole) in 40 ml ofmethanol was cooled to ice-bath temperature, and then 2.0 g of sodiumborohydride was added portionwise, with stirring. Stirring was continuedfor 1 hour, and then the reaction mixture was diluted with water andethyl acetate. The pH was adjusted to 9, and the ethyl acetate layer wasremoved, washed with water, and dried. Evaporation of the ethyl acetatesolution afforded 500 mg of the 9a-deoxo-9a-aza-9a-homo compound.

The latter 9a-deoxo-9a-aza-9a-homo compound was combined with anadditional 1.28 g of material of similar quality prepared in ananalogous fashion, and the mixture was purified by column chromatographyon silica gel, using chloroform/methanol/ammonium hydroxide (9:5:0.05)as eluant. Evaporation of the appropriate fractions gave 600 mg of9-deoxo-4"-deoxy-4"-alpha-amino-9a-aza-9a-homoerythromycin A.

The ¹ H-NMR spectrum of the product showed absorptions at 2.29 (singlet,6H) and 3.33 (singlet, 3H) ppm.

EXAMPLE 4 9-Deoxo-4"-deoxy-4"-beta-amino-9a-aza-9a-homoerythromycin A A.4"-Deoxy-4"-beta-aminoerythromycin A oxime

A mixture of 50 g (68 mmole) of 4"-deoxy-4"-beta-aminoerythromycin A, 25g (360 mmole) of hydroxylamine hydrochloride and 250 ml of pyridine wasstirred at room temperature for 2 days. To the reaction mixture was thenadded water and ethyl acetate and the pH was adjusted to 10. The ethylacetate layer was removed, dried, and evaporated in vacuo to give afoam. The foam was recrystallized from ether to give 14 g of the desiredoxime. The mother liquor was evaporated in vacuo to give a foam, whichwas triturated under petroleum ether and then recrystallized from etherto give an additional 6.0 g of the desired oxime.

Evaporation of the mother liquor from the second recrystallization, andtreatment of the residue with 15 g of hydroxylamine hydrochloride in 60ml of pyridine as above, afforded a further 4.5 g of the desired oxime.

B. 4"-Deoxy-4"-beta-amino-9a-aza-9a-homoerythromycin A

A mixture was prepared from 14.0 g (18.7 mmole) of4"-deoxy-4"-beta-aminoerythromycin A from Part A, 5.3 g (28 mmole) of4-toluenesulfonyl chloride, 4.2 ml (30 mmole) of triethylamine and 100ml of chloroform, with stirring. The temperature rose to 33° C., andthen the reaction mixture was cooled to room temperature with ice-bathcooling. The reaction mixture was stirred at room temperature for 1hour, and then an excess of water was added. The pH of the aqueous phasewas adjusted to 5 using 1N hydrochloric acid and the layers wereseparated. The pH of the aqueous layer was adjusted to 10, and then itwas extracted with ethyl acetate. The ethyl acetate extracts were driedand evaporated in vacuo. The residue was recrystallized from ether togive 7.0 g of the desired 9a -aza-9a-homo compound.

C. 9-Deoxo-4"-deoxy-4"-beta-amino-9a-aza-9a-homoerythromycin A

The 4"-deoxy-4"-beta-amino-9a-aza-9a-homoerythromycin A from Step B (7.0g; 9.4 mmole) was dissolved in 300 ml of methanol and cooled to 10°-15°C. in an ice-bath. To this solution was then added 7.5 g (0.2 mole) ofsodium borohydride portionwise with stirring, during about 20 minutes.Stirring was continued for 3 hours and then an excess of water wasadded. The resulting mixture was extracted several times with chloroformand the extracts were dried and evaporated in vacuo. The residue (9.0 g)was dissolved in a mixture of 100 ml of acetone and 50 ml of water, and9.0 g (55 mmole) of mannitol was added, followed by 1.7 g (20 mmole) ofsodium bicarbonate. The resulting mixture was stirred at roomtemperature for 18 hours, and then it was diluted with water andextracted with ethyl acetate. The ethyl acetate solution was dried andevaporated to give 1.5 g of a first crop of the desired9-deoxo-9a-aza-9a-homo compound.

The residual aqueous phase from the ethyl acetate extraction was furtherextracted with chloroform, and the chloroform solution was dried andevaporated to give 4.5 g of residue. The residue was dissolved in 300 mlof chloroform, and 150 g of silica gel was added. The mixture wasstirred at room temperature for 18 hours, and then it was filtered. Thesilica gel was washed with 300 ml of chloroform, followed by 500 ml ofchloroform/methanol/ammonium hydroxide (100:1:0.1), followed by 500 mlof chloroform/methanol/ammonium hydroxide (4:1:0.1). The originalfiltrate, after removal of the silica gel, and all of the silica gelwashings were combined and evaporated in vacuo. The residue was combinedwith the first crop of the desired 9-deoxo-9a-aza-9a-homo compound fromabove and added to 100 ml of water. The pH was adjusted to 5 and themixture was stirred at pH 5 for 25 minutes. The pH was raised to 10, andthe aqueous phase was extracted with dichloromethane. Thedichloromethane extracts were dried and evaporated in vacuo to give 4.3g of 9-deoxo-4"-deoxy-4"-beta-amino-9a-aza-9a-homoerythromycin A.

The ¹ H-NMR spectrum of the product showed absorptions at 2.24 (singlet,6H) and 3.28 (singlet, 3H) ppm.

EXAMPLE 59-Deoxo-9a-methyl-4"-deoxy-4"-alpha-amino-9a-aza-9a-homoerythromycin AA. 9-Deoxo-9a-methyl-4"-deoxy-4"-oxo-9a-aza-9a-homoerythromycin A oxime

A solution of 7.5 g (9.5 mmole) of9-deoxo-9a-methyl-2'-O-acetyl-4"-deoxy-4"-oxo-9a-aza-9a-homoerythromycinA in 50 ml of methanol was stored at room temperature for 2 days, andthen 3.5 g (50 mmole) of hydroxylamine hydrochloride was added. Theresulting mixture was stirred at room temperature for 3 hours and thenthe solvent was removed by evaporation in vacuo. The residue waspartitioned between ethyl acetate and water, and the pH of the aqueousphase was raised to 9. The layers were separated and the organic layerwas dried and evaporated in vacuo. The residue was recrystallized fromether to give 4.4 g of the desired oxime.

B. 9-Deoxo-9a-methyl-4"-deoxy-4"-alpha-amino-9a-aza-9a-homoerythromycinA

A mixture of 4.4 g (5.8 mmole) of the oxime from Part A and ca. 4 g ofRaney nickel in 100 ml of ethanol was shaken under an atmosphere ofhydrogen at a pressure of 4.5 kg/cm² for ca. 75 hours. The mixture wasthen filtered, and the filtrate was evaporated in vacuo to give a foam.The foam was dissolved in diisopropyl ether, and the solvent was allowedto evaporate slowly. After 24 hours, the white solid which hadprecipitated was collected to give 2.2 g of9-deoxy-9a-methyl-4"-deoxy-4"-alpha-amino-9a-aza-9a-homoerythromycin A.

The ¹ H-NMR spectrum of the product showed absorptions at 2.31 (singlet,6H), 2.35 (singlet, 3H) and 3.31 (singlet, 3H) ppm.

EXAMPLE 69-Deoxo-9a-methyl-4"-deoxy-4"-beta-amino-9a-aza-9a-homoerythromycin A A.9-Deoxo-9a-methyl-4"-deoxy-4"-oxo-9a-aza-9a-homoerythromycin A

A solution of 0.93 g (1.2 mmole) of9-deoxo-9a-methyl-2'-O-acetyl-4"-deoxy-4"-oxo-9a-aza-9a-homoerythromycinA in 50 ml of methanol was stored at room temperature for 20 hours, andthen the solvent was removed by evaporation in vacuo. This afforded 0.74g of the desired deacetylated material.

The ¹ H-NMR spectrum of the product showed absorptions at 2.30 (singlet,6H), 2.38 (singlet, 3H) and 3.35 (singlet, 3H) ppm.

B. 9-Deoxo-9a-methyl-4"-deoxy-4"-beta-amino-9a-aza-9a-homoerythromycin A

A solution of 0.50 g (0.67 mmole) of9-deoxo-9a-methyl-4"-deoxy-4"-oxo-9a-aza-9a-homoerythromycin A and 0.54g (6.7 mmole) of ammonium acetate in 50 ml of methanol was prepared, andthen acetic acid (7 drops) was added with stirring to adjust the pH to6. Stirring was continued for 1 hour, and then 0.13 g (2.1 mmole) ofsodium cyanoborohydride was added portionwise. Stirring was continued anadditional 2.5 hours, and then the reaction mixture was evaporated invacuo. The residue was partitioned between chloroform and water and thepH of the water layer was adjusted to 2. The aqueous layer was removedand the pH was adjusted to 6.2. The aqueous layer was extracted at pH6.2 to remove the 4"-hydroxy products and the extracts were discarded.The pH of the aqueous layer was then raised to 9.5 and the aqueous layerwas then further extracted with chloroform. The latter extracts weredried and evaporated in vacuo. The residue was redissolved in water atpH 2. The aqueous solution thus obtained was extracted with chloroformat pH 2, pH 6.2 and pH 9.5. The chloroform solution from the extractionat pH 9.5 was dried, evaporated and again dissolved in water at pH 2.This latter aqueous solution was extracted with chloroform at pH 2, pH6.2 and pH 9.5. The chloroform solution from the extraction at pH 9.5was dried and evaporated in vacuo to give 0.18 g of a 1:1 mixture of9-deoxo-9a-methyl-4"-deoxy-4"-beta-amino-9a-aza-9a-homoerythromycin Aand its 4"-alpha-epimer.

EXAMPLE 79-Deoxo-9a-methyl-2'-O-acetyl-4"-deoxy-4"-oxo-9a-aza-9a-homoerythromycinA A. 9-Deoxo-9a-methyl-2'-O-acetyl-9a-aza-9a-homoerythromycin A

A solution was prepared from 1.0 g (1.3 mmole) of9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A, and 0.13 ml (1.4 mmole)of acetic anhydride in 15 ml of chloroform was stirred for several hoursat room temperature. To the solution was added an excess of water andstirring was continued for 30 minutes with the pH being held at 9. Theorganic phase was then removed, dried and evaporated to give 1.0 g ofthe desired 2'-O-acetyl compound.

B.9-Deoxo-9a-methyl-2'-O-acetyl-4"-deoxy-4"-oxo-9a-aza-9a-homoerythromycinA

A mixture was prepared from 7.5 g (9.5 mmole) of9-deoxy-9a-methyl-2'-O-acetyl-9a-aza-9a-homoerythromycin A, 5.5 g (28mmole) of N-ethyl-N'-(N,N-dimethylaminopropyl)carbodiimide and 6.7 ml(95 mmole) of dimethyl sulfoxide in 75 ml of dichloromethane. To thismixture was then added, dropwise, with stirring, during 3 minutes, 5.5 g(28 mmole) of pyridinium trifluoroacetate. The temperature rose to 39°C. and then returned to room temperature. Stirring was continued for 2hours and then an excess of water was added and the pH of the aqueouslayer was adjusted to 9. The organic layer was removed, dried andevaporated in vacuo to give 7.5 g of9-deoxo-9a-methyl-2'-O-acetyl-4"-deoxy-4"-oxo-9a-aza-9a-homoerythromycinA.

The ¹ H-NMR spectrum of the product showed absorptions at 2.05 (singlet,3H), 2.26 (singlet, 6H), 2.33 (singlet, 3H) and 3.33 (singlet, 3H) ppm.

EXAMPLE 89-Deoxo-9a-methyl-2'-O-propionyl-4"-deoxy-4"-oxo-9a-aza-9a-homoerythromycinA

The title compound can be prepared by repeating Example 7, but replacingthe acetic anhydride used in Part A by an equimolar amount of propionicanhydride.

PREPARATION 1 4"-Deoxy-4"-alpha-amino-erythromycin A

A mixture of 10.0 g (13.6 mmole) of 4"-deoxy-4"-oxo-erythromycin A, 10.5g of ammonium acetate and 10.0 g of Raney nickel in 150 ml of methanolwas shaken under an atmosphere of hydrogen, at an initial hydrogenpressure of ca. 4 kg/cm², at room temperature, overnight. An additional10.5 g of ammonium acetate and 10.0 g of Raney nickel were then addedand the mixture was again shaken under hydrogen, at initial hydrogenpressure of ca. 4 kg/cm², at room temperature, overnight. The catalystwas removed by filtration and the filtrate was concentrated in vacuo toca. 50 ml. The concentrated filtrate was then poured with stirring intoa mixture of 250 ml of water and 200 ml of chloroform, and the pH of theaqueous layer was adjusted to 5.4. The organic layer was removed anddiscarded and the aqueous layer was further extracted with chloroform atpH 5.4. The further extracts were discarded. The pH of the aqueous phasewas adjusted to 9.6 and then the aqueous phase was extracted withchloroform. The latter extracts were dried (Na₂ SO₄) and thenconcentrated in vacuo to give 5.74 g of a white foam. The foam wasdissolved in 35 ml of hot isopropanol, and the solution was allowed tocool to room temperature with stirring. The solid which had formed wasrecovered by filtration and dried, to afford 3.54 g of4"-deoxy-4"-alpha-amino-erythromycin A, contaminated with 5-10% of its4"-epimer.

The proportion of 4"-beta-amino epimer can be reduced by successiverecrystallizations from isopropanol.

PREPARATION 2 4"-Deoxy-4"-beta-amino-erythromycin A

Twenty grams of 4"-deoxy-4"-oxo-erythromycin A, 31.6 g of ammoniumacetate and 10 g of 10% palladium-on-carbon in 200 ml of methanol wasshaken at ambient temperatures in a hydrogen atmosphere at an initialpressure of ca. 4 kg/cm² overnight. The spent catalyst was filtered andthe filtrate concentrated to dryness in vacuo. The residue waspartitioned between water-chloroform at a pH of 5.5. The aqueous layerwas separated, the pH adjusted to 9.6 and chloroform added. The organiclayer was separated, dried over sodium sulfate and concentrated underreduced pressure to dryness. The residual white foam (19 g) wastriturated with 150 ml of diethyl ether at room temperature for 30minutes. The resulting solids were filtered and dried to give 9.45 g of4"-deoxy-4"-beta-amino-erythromycin A.

We claim:
 1. A macrolide antibiotic compound of the formula ##STR10##and the pharmaceutically-acceptable acid-addition salts thereof; whereinR¹ is selected from the group consisting of hydrogen and methyl; andR²and R³ are each selected from the group consisting of hydrogen andamino; provided that R² and R³ are always different.
 2. A compoundaccording to claim 1, wherein R¹ is methyl.
 3. The compound according toclaim 2, wherein R² is amino and R³ is hydrogen.
 4. The compoundaccording to claim 2, wherein R² is hydrogen and R³ is amino.
 5. Amethod of treating a bacterial infection in a mammalian subject, whichcomprises administering thereto an antibacterially effective amount of amacrolide antibiotic compound of the formula ##STR11## or apharmaceutically-acceptable acid-addition salt thereof; wherein R¹ isselected from the group consisting of hydrogen and methyl; andR² and R³are each selected from the group consisting of hydrogen and amino;provided that R² and R³ are always different.
 6. The method according toclaim 5, wherein R¹ is methyl.
 7. The method according to claim 6,wherein R² is amino and R³ is hydrogen.
 8. The method according to claim6, wherein R² is hydrogen and R³ is amino.
 9. A pharmaceuticalcomposition which comprises a pharmaceutically-acceptable carrier and amacrolide antibiotic compound according to claim 1, wherein the weightratio of the pharmaceutically-acceptable carrier to the macrolideantibiotic compound is in the range from 4:1 to 1:4.
 10. A compound ofthe formula ##STR12## and the acid-addition salts thereof, wherein R⁴ isselected from the group consisting of hydrogen, acetyl and propionyl.11. The compound according to claim 10, wherein R⁴ is hydrogen.
 12. Thecompound according to claim 10, wherein R⁴ is acetyl.